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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

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Ed. by Gillery, Philippe / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Payne, Deborah A. / Schlattmann, Peter / Tate, Jillian R.

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Volume 56, Issue 1


Determination of reference intervals for urinary steroid profiling using a newly validated GC-MS/MS method

Wilhelmina H.A. de Jong
  • Corresponding author
  • Department of Laboratory Medicine, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands
  • Email
  • Other articles by this author:
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/ Edward Buitenwerf
  • Department of Endocrinology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Alle T. Pranger
  • Department of Laboratory Medicine, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Ineke J. RiphagenORCID iD: http://orcid.org/0000-0001-5500-0145 / Bruce H.R. Wolffenbuttel
  • Department of Endocrinology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Michiel N. Kerstens
  • Department of Endocrinology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands
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  • De Gruyter OnlineGoogle Scholar
/ Ido P. Kema
  • Department of Laboratory Medicine, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
Published Online: 2017-06-09 | DOI: https://doi.org/10.1515/cclm-2016-1072



Urinary steroid profiling (USP) is a powerful diagnostic tool to asses disorders of steroidogenesis. Pre-analytical factors such as age, sex and use of oral contraceptive pills (OCP) may affect steroid hormone synthesis and metabolism. In general, USP reference intervals are not adjusted for these variables. In this study we aimed to establish such reference intervals using a newly-developed and validated gas chromatography with tandem mass spectrometry detection method (GC-MS/MS).


Two hundred and forty healthy subjects aged 20–79 years, stratified into six consecutive decade groups each containing 20 males and 20 females, were included. None of the subjects used medications. In addition, 40 women aged 20–39 years using OCP were selected. A GC-MS/MS assay, using hydrolysis, solid phase extraction and double derivatization, was extensively validated and applied for determining USP reference intervals.


Androgen metabolite excretion declined with age in both men and women. Cortisol metabolite excretion remained constant during life in both sexes but increased in women 70–79 years of age. Progesterone metabolite excretion peaked in 30–39-year-old women and declined afterwards. Women using OCP had lower excretions of androgen metabolites, progesterone metabolites and cortisol metabolites. Method validation results met prerequisites and revealed the robustness of the GC-MS/MS method.


We developed a new GC-MS/MS method for USP which is applicable for high throughput analysis. Widely applicable age and sex specific reference intervals for 33 metabolites and their diagnostic ratios have been defined. In addition to age and gender, USP reference intervals should be adjusted for OCP use.

This article offers supplementary material which is provided at the end of the article.

Keywords: GC-MS/MS; oral contraceptive pills; reference intervals; urinary steroid profile


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About the article

Corresponding author: Wilhelmina H.A. de Jong, PhD, Department of Laboratory Medicine, University of Groningen, University Medical Center Groningen, Hanzeplein 1, EA61, PO-Box 30.001, 9700 RB Groningen, The Netherlands, Phone: +0031-50-3617015, Fax: +050-3611746

Received: 2016-11-24

Accepted: 2017-04-20

Published Online: 2017-06-09

Published in Print: 2017-11-27

Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

Research funding: None declared.

Employment or leadership: None declared.

Honorarium: None declared.

Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

Citation Information: Clinical Chemistry and Laboratory Medicine (CCLM), Volume 56, Issue 1, Pages 103–112, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/cclm-2016-1072.

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